Certain products and by-products of naturally-occurring metabolic processes in cells have utility in a wide array of industries, including the food, feed, cosmetics, and pharmaceutical industries. These molecules, collectively termed xe2x80x98fine chemicalsxe2x80x99, include organic acids, both proteinogenic and non-proteinogenic amino acids, nucleotides and nucleosides, lipids and fatty acids, diols, carbohydrates, aromatic compounds, vitamins and cofactors, and enzymes. Their production is most conveniently performed through large-scale culture of bacteria developed to produce and secrete large quantities of a particular desired molecule. One particularly useful organism for this purpose is Corynebacterium glutamicum, a gram positive, nonpathogenic bacterium. Through strain selection, a number of mutant strains have been developed which produce an array of desirable compounds. However, selection of strains improved for the production of a particular molecule is a time-consuming and difficult process.
The invention provides novel bacterial nucleic acid molecules which have a variety of uses. These uses include the identification of microorganisms which can be used to produce fine chemicals, the modulation of fine chemical production in C. glutamicum or related bacteria, the typing or identification of C. glutamicum or related bacteria, as reference points for mapping the C. glutamicum genome, and as markers for transformation. These novel nucleic acid molecules encode proteins, referred to herein as stress, resistance and tolerance (SRT) proteins.
C. glutamicum is a gram positive, aerobic bacterium which is commonly used in industry for the large-scale production of a variety of fine chemicals, and also for the degradation of hydrocarbons (such as in petroleum spills) and for the oxidation of terpenoids. The SRT nucleic acid molecules of the invention, therefore, can be used to identify microorganisms which can be used to produce fine chemicals, e.g, by fermentation processes. Modulation of the expression of the SRT nucleic acids of the invention, or modification of the sequence of the SRT nucleic acid molecules of the invention, can be used to modulate the production of one or more fine chemicals from a microorganism (e.g. to improve the yield or production of one or more fine chemicals from a Corynebacterium or Brevibacterium species).
The SRT nucleic acids of the invention may also be used to identify an organism as being Corynebacterium glutamicum or a close relative thereof, or to identify the presence of C. glutamicum or a relative thereof in a mixed population of microorganisms. The invention provides the nucleic acid sequences of a number of C glutamicum genes; by probing the extracted genomic DNA of a culture of a unique or mixed population of microorganisms under stringent conditions with a probe spanning a region of a C. glutamicum gene which is unique to this organism, one can ascertain whether this organism is present. Although Corynebacterium glutamicum itself is nonpathogenic, it is related to species pathogenic in humans, such as Corynebacterium diphtheriae (the causative agent of diphtheria); the detection of such organisms is of significant clinical relevance.
The SRT nucleic acid molecules of the invention may also serve as reference points for mapping of the C. glutamicum genome, or of genomes of related organisms. Similarly, these molecules, or variants or portions thereof, may serve as markers for genetically engineered Corynebacterium or Brevibacterium species.
The SRT proteins encoded by the novel nucleic acid molecules of the invention are capable of, for example, permitting C. glutamicum to survive in a setting which is either chemically or environmentally hazardous to this microorganism. Given the availability of cloning vectors for use in Corynebacterium glutamicum, such as those disclosed in Sinskey et al., U.S. Pat. No. 4,649,119, and techniques for genetic manipulation of C. glutamicum and the related Brevibacterium species (e.g., lactofermentum) (Yoshihama et al, J. Bacteriol. 162: 591-597 (1985); Katsumata et al., J. Bacteriol. 159: 306-311 (1984); and Santamaria et al, J. Gen. Microbiol. 130: 2237-2246 (1984)), the nucleic acid molecules of the invention may be utilized in the genetic engineering of this organism to make it a better or more efficient producer of one or more fine chemicals, through the ability of these proteins to permit growth and multiplication of C. glutamicum (and also continuous production of one or more fine chemicals) under circumstances which would normally impede growth of the organism, such as those conditions frequently encountered during large-scale fermentative growth. For example, by overexpressing or engineering a heat-shock induced protease molecule such that it is optimized in activity, one may increase the ability of the bacterium to degrade incorrectly folded proteins when the bacterium is challenged with high temperatures. By having fewer misfolded (and possibly misregulated or nonfunctional) proteins to interfere with normal reaction mechanisms in the cell, the cell is increased in its ability to function normally in such a culture, which should in turn provide increased viability. This overall increase in number of cells having greater viability and activity in the culture should also result in an increase in yield, production, and/or efficiency of production of one or more desired fine chemicals, due at least to the relatively greater number of cells producing these chemicals in the culture.
This invention provides novel SRT nucleic acid molecules which encode SRT proteins which are capable of, for example, permitting C. glutamicum to survive in a setting which is either chemically or environmentally hazardous to this microorganism. Nucleic acid molecules encoding an SRT protein are referred to herein as SRT nucleic acid molecules. In a preferred embodiment, the SRT protein participates in metabolic pathways permitting C. glutamicum to survive in a setting which is either chemically or environmentally hazardous to this microorganism. Examples of such proteins include those encoded by the genes set forth in Table 1.
Accordingly, one aspect of the invention pertains to isolated nucleic acid molecules (e.g., cDNAs, DNAs, or RNAs) comprising a nucleotide sequence encoding an SRT protein or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection or amplification of SRT-encoding nucleic acid (e.g., DNA or mRNA). In particularly preferred embodiments, the isolated nucleic acid molecule comprises one of the nucleotide sequences set forth in Appendix A or the coding region or a complement thereof of one of these nucleotide sequences. In other particularly preferred embodiments, the isolated nucleic acid molecule of the invention comprises a nucleotide sequence which hybridizes to or is at least about 50%, preferably at least about 60%, more preferably at least about 70%, 80% or 90%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to a nucleotide sequence set forth in Appendix A, or a portion thereof. In other preferred embodiments, the isolated nucleic acid molecule encodes one of the amino acid sequences set forth in Appendix B. The preferred SRT proteins of the present invention also preferably possess at least one of the SRT activities described herein.
In another embodiment, the isolated nucleic acid molecule encodes a protein or portion thereof wherein the protein or portion thereof includes an amino acid sequence which is sufficiently homologous to an amino acid sequence of Appendix B, e.g., sufficiently homologous to an amino acid sequence of Appendix B such that the protein or portion thereof maintains an SRT activity. Preferably, the protein or portion thereof encoded by the nucleic acid molecule maintains the ability to increase the survival of C. glutamicum in a setting which is either chemically or environmentally hazardous to this microorganism. In one embodiment, the protein encoded by the nucleic acid molecule is at least about 50%, preferably at least about 60%, and more preferably at least about 70%, 80%, or 900% and most preferably at least about 95%, 96%, 97%, 98%, or 99% or more homologous to an amino acid sequence of Appendix B (e.g., an entire amino acid sequence selected from those sequences set forth in Appendix B). In another preferred embodiment, the protein is a full length C. glutamicum protein which is substantially homologous to an entire amino acid sequence of Appendix B (encoded by an open reading frame shown in Appendix A).
In another preferred embodiment, the isolated nucleic acid molecule is derived from C. glutamicum and encodes a protein (e.g., an SRT fusion protein) which includes a biologically active domain which is at least about 50% or more homologous to one of the amino acid sequences of Appendix B and has the ability to increase the survival of C. glutamicum in a setting which is either chemically or environmentally hazardous to this microorganism, or possesses one or more of the activities set forth in Table 1, and which also includes heterologous nucleic acid sequences encoding a heterologous polypeptide or regulatory regions.
In another embodiment, the isolated nucleic acid molecule is at least 15 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising a nucleotide sequence of Appendix A. Preferably, the isolated nucleic acid molecule corresponds to a naturally-occurring nucleic acid molecule. More preferably, the isolated nucleic acid encodes a naturally-occurring C. glutamicum SRT protein, or a biologically active portion thereof.
Another aspect of the invention pertains to vectors, e.g., recombinant expression vectors, containing the nucleic acid molecules of the invention, and host cells into which such vectors have been introduced. In one embodiment, such a host cell is used to produce an SRT protein by culturing the host cell in a suitable medium. The SRT protein can be then isolated from the medium or the host cell.
Yet another aspect of the invention pertains to a genetically altered microorganism in which an SRT gene has been introduced or altered. In one embodiment, the genome of the microorganism has been altered by the introduction of a nucleic acid molecule of the invention encoding wild-type or mutated SRT sequence as a transgene. In another embodiment, an endogenous SRT gene within the genome of the microorganism has been altered, e.g., functionally disrupted, by homologous recombination with an altered SRT gene. In another embodiment, an endogenous or introduced SRT gene in a microorganism has been altered by one or more point mutations, deletions, or inversions, but still encodes a functional SRT protein. In still another embodiment, one or more of the regulatory regions (e.g., a promoter, repressor, or inducer) of a SRT gene in a microorganism has been altered (e.g. by deletion, truncation, inversion, or point mutation) such that the expression of the SRT gene is modulated. In a preferred embodiment, the microorganism belongs to the genus Corynebacterium or Brevibacterium, with Corynebacterium glutamicum being particularly preferred. In a preferred embodiment, the microorganism is also utilized for the production of a desired compound, such as an amino acid, with lysine being particularly preferred.
In another aspect, the invention provides a method of identifying the presence or activity of Cornyebacterium diphtheriae in a subject. This method includes detection of one or more of the nucleic acid or amino acid sequences of the invention (e.g., the sequences set forth in Appendix A or Appendix B) in a subject, thereby detecting the presence or activity of Corynebacterium diphtheriae in the subject.
Still another aspect of the invention pertains to an isolated SRT protein or a portion, e.g., a biologically active portion, thereof. In a preferred embodiment, the isolated SRT protein or portion thereof possesses the ability to increase the survival of C. glutamicum in a setting which is either chemically or environmentally hazardous to this microorganism. In another preferred embodiment, the isolated SRT protein or portion thereof is sufficiently homologous to an amino acid sequence of Appendix B such that the protein or portion thereof maintains the ability to increase the survival of C. glutamicum in a setting which is either chemically or environmentally hazardous to this microorganism.
The invention also provides an isolated preparation of an SRT protein. In preferred embodiments, the SRT protein comprises an amino acid sequence of Appendix B. In another preferred embodiment, the invention pertains to an isolated full length protein which is substantially homologous to an entire amino acid sequence of Appendix B (encoded by an open reading frame set forth in Appendix A). In yet another embodiment, the protein is at least about 50%, preferably at least about 60%, and more preferably at least about 70%, 80%, or 90%, and most preferably at least about 95%, 96%, 97%, 98%, or 99% or more homologous to an entire amino acid sequence of Appendix B. In other embodiments, the isolated SRT protein comprises an amino acid sequence which is at least about 50% or more homologous to one of the amino acid sequences of Appendix B and is able to improve the survival rate of C. glutamicum in a setting which is either chemically or environmentally hazardous to this microorganism, or has one or more of the activities set forth in Table 1.
Alternatively, the isolated SRT protein can comprise an amino acid sequence which is encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, or is at least about 50%, preferably at least about 60%, more preferably at least about 70%, 80%, or 90%, and even more preferably at least about 95%, 96%, 97%, 98,%, or 99% or more homologous, to a nucleotide sequence of Appendix B. It is also preferred that the preferred forms of SRT proteins also have one or more of the SRT bioactivities described herein.
The SRT polypeptide, or a biologically active portion thereof, can be operatively linked to a non-SRT polypeptide to form a fusion protein. In preferred embodiments, this fusion protein has an activity which differs from that of the SRT protein alone. In other preferred embodiments, this fusion protein results in increased yields, production, and/or efficiency of production of a desired fine chemical from C. glutamicum. In particularly preferred embodiments, integration of this fusion protein into a host cell modulates the production of a desired compound from the cell.
In another aspect, the invention provides methods for screening molecules which modulate the activity of an SRT protein, either by interacting with the protein itself or a substrate or binding partner of the SRT protein, or by modulating the transcription or translation of an SRT nucleic acid molecule of the invention.
Another aspect of the invention pertains to a method for producing a fine chemical. This method involves the culturing of a cell containing a vector directing the expression of an SRT nucleic acid molecule of the invention, such that a fine chemical is produced. In a preferred embodiment, this method further includes the step of obtaining a cell containing such a vector, in which a cell is transfected with a vector directing the expression of an SRT nucleic acid. In another preferred embodiment, this method further includes the step of recovering the fine chemical from the culture. In a particularly preferred embodiment, the cell is from the genus Corynebacterium or Brevibacterium, or is selected from those strains set forth in Table 3.
Another aspect of the invention pertains to methods for modulating production of a molecule from a microorganism. Such methods include contacting the cell with an agent which modulates SRT protein activity or SRT nucleic acid expression such that a cell associated activity is altered relative to this same activity in the absence of the agent. In a preferred embodiment, the cell is modulated in resistance to one or more toxic chemicals or in resistance to one or more environmental stresses, such that the yields or rate of production of a desired fine chemical by this microorganism is improved. The agent which modulates SRT protein activity can be an agent which stimulates SRT protein activity or SRT nucleic acid expression. Examples of agents which stimulate SRT protein activity or SRT nucleic acid expression include small molecules, active SRT proteins, and nucleic acids encoding SRT proteins that have been introduced into the cell. Examples of agents which inhibit SRT activity or expression include small molecules, and antisense SRT nucleic acid molecules.
Another aspect of the invention pertains to methods for modulating yields of a desired compound from a cell, involving the introduction of a wild-type or mutant SRT gene into a cell, either maintained on a separate plasmid or integrated into the genome of the host cell. If integrated into the genome, such integration can random, or it can take place by homologous recombination such that the native gene is replaced by the introduced copy, causing the production of the desired compound from the cell to be modulated. In a preferred embodiment, said yields are increased. In another preferred embodiment, said chemical is a fine chemical. In a particularly preferred embodiment, said fine chemical is an amino acid. In especially preferred embodiments, said amino acid is L-lysine.